Human leukocyte elastase (HLE, EC.3.4.21.37) is a serine proteinase that is synthesized and processed as a single molecular protein that is targeted exclusively for the cell surface (HLE-CS) early in ontogeny of lymphoid and myeloid cells when they are too immature to form granules. However, HLE is targeted for granule compartmentalization (HLE-G) later in ontogeny when cells develop the capability to form granules (Gullberg et al., 1995; Garwicz et al., 2005). Whereas HLE-G has enzymatic activity, HLE-CS acts as a receptor, and there is no evidence of its having enzymatic activity.
The primary physiologic mechanism for controlling the enzymatic activity of HLE-G is the abundant proteinase inhibitor alpha-1 proteinase inhibitor (α1PI, α1 antitrypsin). When bound to HLE-G, a covalent-like complex is formed in which neither α1PI, nor HLE-G are cleaved (Dementiev et al., 2006). Similarly, when α1PI binds to HLE-CS, a complex is formed which does not appear to involve completion of enzymatic activity, and the complex induces polarization of functionally-related receptors and cell motility (Wolf et al., 2003). Functionally-related receptors include the chemokine receptor CXCR4 (CD184), CD4, and the T cell antigen receptor (TcR).
In healthy individuals, 98% of α1PI is in the active form. Normal ranges are 18-53 μM active and 0-11 μM inactive α1PI (Bristow et al., 2001). Active α1PI circulates in blood in two isoforms in dynamic equilibrium: 1) native α1PI, which binds irreversibly to HLE-CS, and 2) thiol-modified α1PI, which binds reversibly to HLE-CS (Tyagi, 1991).
Inactive α1PI arises during infection or inflammation via modification of active α1PI by factors released from microorganisms or host cells. Inactive α1PI can arise by complexing with HLE-G or HLE-CS, being cleaved by proteinases other than HLE, or oxygenation. In its inactivated form, α1PI binds to low density lipoprotein (LDL), apoB, and members of the LDL receptor family (LDL-RFMs), whereby α1PI facilitates LDL uptake into cells (Mashiba et al., 2001; Janciauskiene et al., 2001).
Binding of active α1PI to HLE-CS at the leading edge of migrating cells induces aggregation and polarization of LDL-RFMs with other functionally-related receptors (Bristow et al., 2003; 2008; Bristow and Flood, 1993). Cellular locomotion repositions the HLE-CS complex including functionally-associated receptors to the trailing edge of the cell where LDL-RFMs on the same cell bind to the α1PI-HLE-CS complexes through an interaction that involves the α1PI C-terminal domain (C-36, VIRIP) (Kounnas et al., 1996; Janciauskiene el al., 2001). This interaction induces internalization (endocytosis) of LDL-RFMs including functionally-related receptors within the aggregate and entities bound to them such as lipoproteins and viruses. This action furthers retraction of the trailing edge of the migrating cell thereby promoting forward locomotion (Kounnas el al., 1996; Cao el al., 2006; Bristow et al., 2003; Bristow et al., 2013).
The recycling of endocytosed receptors and polarization at the leading edge of a migrating cells followed by endocytosis at the trailing edge operates somewhat like a conveyor belt. If one of the components involved in this conveyor belt mechanism is missing or blocked, the cell halts migrating. For example, bacteria, snake bites, blood clotting, and most other non-normal situations produce non-normal proteases which cleave sentinel proteinase inhibitors including α1PI. When α1PI is inactivated, it can no longer bind its receptor HLE-CS. In the absence of α1PI-HLE-CS complexes, the LDL-RFMs are not triggered for endocytosis and this causes blood cells to stop migrating. This mechanistic process provides a method for locomoting immune cells to sample the environment for nutrients (e.g. lipoproteins and insulin-coupled glucose), toxic material (e.g. viruses, bacterial enzymes, inflammatory products), or inert material (e.g. recycled receptors). Due to the dynamics of the process, targeting HLE-CS using levorotatory β-lactams allows regulation of hematopoiesis, lipoprotein levels, and unwanted tissue degradation.
Cell motility results from selective and sequential adherence and release produced by activation and deactivation of receptors (Wright and Meyer, 1986; Ali et al., 1996), consequent polar segregation of related membrane proteins to the leading edge or trailing uropod, and both clockwise and counterclockwise propagation of Ca++ waves which initiate from different locations in the cell (Kindzelskii and Petty, 2003). Thus, several aspects of the complex process may be quantitated. The most direct and most easily interpreted method for quantitating cell motility is the enumeration of adherent cells in response to a chemotactic agent such as α1PI.
Applicant's co-pending U.S. patent application Ser. No. 13/302,821 is directed to a method for increasing the number of circulating CD4+ T-lymphocytes in subjects receiving antiretroviral therapy comprising administering to a subject in need of such treatment an amount of active α1PI effective to increase the number of circulating CD4+ T-lymphocytes in the subject.
Applicant's co-pending Ser. No. 13/948,446 is directed to a method of modulating LDL levels, HDL levels, cholesterol levels, and triglyceride levels in a subject comprising administering to the subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of active α1PI, thereby modulating the distribution of LDL levels, HDL levels, cholesterol levels, and triglyceride levels in the subject.
The hematopoietic role of α1PI has not been therapeutically exploited thus far. Its use has been limited due to its high cost and short supply. Previous therapeutic applications of α1PI have been restricted to augmentation for the purpose of ameliorating respiratory distress such as occurs in emphysema and chronic obstructive pulmonary disease (COPD) in patients diagnosed with inherited α1PI deficiency. Commercially, α1PI is available from a few sources including PROLASTIN®-C, (Grifols Therapeutics Inc., Research Triangle Park, N.C.) and ZEMAIRA® (CSL Behring LLC, King of Prussia, Pa.) as a cryoprecipitate preparation isolated from human plasma.
What is needed in the art are small molecules to act as surrogates for α1PI. The present invention provides such small molecule compounds useful in arresting degradative enzymatic activity for the treatment of respiratory distress such as occurs in emphysema and COPD, modulating lipoprotein and apolipoprotein levels, and for regulating the number of circulating CD4+ or CD8+ T-lymphocytes (CD4/CD8 ratio) in subjects in need of such treatment.